Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.     

Estrogen and progesterone receptors and cyclooxygenase-2 expression in endometrial cancer, endometrial hyperplasia, and normal endometrium

OBJECTIVES: To determine whether cyclooxygenase-2 (COX-2) expression is seen in endometrial cancer, endometrial hyperplasia, and normal endometria and whether it correlates with expression of estrogen and progesterone receptors. METHODS: The study was a retrospective, IRB-approved analysis of biopsy samples from 14 patients with endometrial adenocarcinoma, 19 with endometrial hyperplasias, and 10 with normal endometrium. Excluded were samples from women with a history of pelvic radiation, NSAID use, or treatment with hormones during previous year. Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tissues. Expression of COX-2, estrogen and progesterone receptors were scored according to the proportion of positive-staining cells: 1(+), <10%; 2(+), 10-50%; and 3(+), >50%. A score > or =2(+) was considered positive. Fisher's exact test and analysis of variance were used to compare proportions and continuous variables, respectively. RESULTS: Overexpression of COX-2 was seen in 4 (29%) of the endometrial cancers, 6 (32%) of the endometrial hyperplasia, and 4 (20%) of the normal endometria. These differences were not statistically significant (P = 0.90). No COX-2 expression was found in stromal tissue. Of 14 endometrial cancers, 7 (50%) expressed any COX-2, with 4 (29%) having an expression score of > or =2(+). Of 19 endometrial hyperplasias, 11 (58%) expressed any COX-2; with 6 (32%) having a score of > or =2(+). All 10 normal endometria showed only 1(+) expression. No significant differences were detected in COX-2 expression by grade or stage of cancer. Although 100% and 95% of both hyperplasia and normal endometrium samples expressed in estrogen and progesterone receptors, respectively, only 71% and 79% of endometrial cancers expressed estrogen and progesterone receptors (P = 0.01). A nonparametric trend was performed to detect a relationship, between COX-2 and estrogen receptor or progesterone receptor expression; no significant trend was found. CONCLUSIONS: In this study, the immunohistochemical analysis showed a trend toward increased COX-2 expression in endometrial cancer and hyperplasia compared to normal endometria. A larger sample size is needed to confirm these results. The increased COX-2 expression in hyperplasia may signify an early step in carcinogenesis. These findings may represent an important treatment opportunity for synergism in the hormonal therapy of endometrial cancer.     

P^21ras c—mycp53和雌孕激素受体在子宫内膜癌的表达

应用免疫组化ＬＳＡＢ法，对３２例子宫内膜癌，６例腺瘤型增生，２０例腺囊型增生组织的癌基因Ｐ＾２１ｒａｓ，ｃ－ｍｙｃ及抑癌基因ｐ５３蛋白表达进行了检测，对３２例子宫内膜癌进行了雌，孕激素受体的检测，并对子宫内膜癌组织Ｐ＾２１ｒａｓ，ｃ－ｍｙｃｐ５３过度表达与雌，孕激素受体的关系了分析，结果显示：Ｐ＾２１ｒａｓｃ－ｍｙｃｐ５３在子宫内膜癌组织中的阳性表达率分别为５９．４％，６２．５％和１８．８％，雌，     

Distinct functional differences of human progesterone receptors A and B on gene expression and growth regulation in two endometrial carcinoma cell lines

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59).Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells.We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.     

Modulation of endometrial steroid receptors and growth regulatory genes by tamoxifen

OBJECTIVE: We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation. METHODS: Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-alpha, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques. RESULTS: Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P <. 003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-alpha, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens. CONCLUSION: These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.     

Molecular tools to reestablish progestin control of endometrial cancer cell proliferation

OBJECTIVE: Endometrial cancers often arise in a setting of estrogen stimulation unopposed by the differentiating effects of progesterone. Our laboratory and others have previously shown that progesterone receptor down-regulation or perturbation of progesterone receptor isoform A or B expression is associated with the development of poorly differentiated endometrial cancers that are not growth inhibited by progestins. The purpose of these studies was to reestablish high progesterone receptor isoform A and B gene expressions in such endometrial cancer cells and to examine the effects of progestin treatment on cell growth and metastatic potential after this transformation. STUDY DESIGN: To induce high levels of expression of the progesterone receptor isoforms in KLE and Hec50 endometrial cancer cells, adenoviral vectors encoding the genes for progesterone receptor isoforms A and B were created. The characteristic ability of cancer cells to grow independently of anchorage to the surrounding solid matrix was measured by counting colony formation on soft agar for 8 to 14 days. Cell proliferation in response to a time course of progestin treatment was tested with flow cytometry. RESULTS: After treatment with a control vector without a progesterone receptor--encoding insert, no effect of progestin treatment on cell proliferation was found; after treatment with vectors encoding progesterone receptor isoform A or B, however, progestin treatment resulted in significant inhibition of cell growth. The anchorage-independent cell growth on soft agar assay showed that by 8 to 14 days the number of cell colonies was reduced by 50% relative to control preparations in the presence of progesterone receptor isoform A plus progestin (P <.0001, both Hec50 and KLE cell lines) and by 90% in the presence of progesterone receptor isoform B plus progestin (P <.0001, both Hec50 and KLE cell lines). Progestin treatment also resulted in a time-dependent reduction in cell proliferation as measured by flow cytometry. Although transfection with both progesterone receptor isoforms A and B reduced cell proliferation according to our assays, progesterone receptor isoform B caused a much more dramatic decrease in cell growth (P =.001, Hec50 cells; P <.0001, KLE cells). CONCLUSION: In poorly differentiated endometrial cancer cells that are resistant to progestin therapy, adenovirus-induced expressions of progesterone receptors A and B reestablish progestin control of endometrial cancer cell proliferation.     

Progesterone-mediated regulation of catechol-O-methyl transferase expression in endometrial cancer cells

The effects of estrogen and progesterone on the expression of estrogen-metabolizing enzymes such as catechol-O-methyl transferase (COMT) are not known. COMT converts genotoxic catecholestrogens to anticarcinogenic methoxyestrogens in the endometrium. The aim of this study is to investigate the effect of progesterone on COMT expression in well-differentiated endometrial cancer cells. The wild-type Ishikawa cell line as well as progesterone receptor A- or progesterone receptor B-transfected Ishikawa cells were used for in vitro studies. The regulation of COMT expression by progesterone was studied using Western blots, Hoechst dye DNA proliferation studies, and wild-type and/or site-directed mutagenesis of COMT promoter 1-luciferase reporter gene. Progesterone upregulated COMT protein expression in Ishikawa cells through progesterone receptor A isoform. COMT promoter activity was differentially regulated by the 3 half-site progesterone response elements in the COMT promoter. High doses of 2-ME2 inhibited Ishikawa cell proliferation. These data suggest that COMT expression is hormonally regulated in well-differentiated human endometrial cancer cells. COMT regulation and 2-ME2 production in the endometrium may affect endometrial carcinogenesis.     

Immunohistochemical expression of TAG-72 in normal and malignant endometrium: correlation of antigen expression with estrogen receptor and progesterone receptor levels

TAG-72 is a tumor-associated antigen that is expressed by secretory endometrium and most endometrial adenocarcinomas. We used immunohistochemical techniques to quantitate expression of TAG-72, estrogen receptor, and progesterone receptor in 21 normal endometria and 44 endometrial adenocarcinomas. In normal cycling endometrial glands, TAG-72 expression was related inversely to expression of receptors for estrogen and progesterone. This suggests that TAG-72 expression in normal endometrium may be hormonally regulated. Ninety-one percent of endometrial adenocarcinomas expressed immunohistochemically detectable TAG-72. The magnitude of TAG-72 expression did not correlate with other known prognostic factors in endometrial cancer such as histologic grade, depth of myometrial invasion, surgical stage, or steroid receptor status. The production of TAG-72 by most endometrial adenocarcinomas may represent nonspecific expression by cells that have dedifferentiated.     

Differential gene expression in progesterone-sensitive and progesterone-insensitive endometrial carcinoma cells

High doses of progesterone are used in the treatment of advanced and recurrent endometrial cancer. Unfortunately the response rate is relatively low: 10-30%. The mechanisms involved in the development of insensitivity to progesterone treatment of endometrial cancer tissue are largely unknown. As tumour development is thought to be associated with a cascade of genetic alterations, it can be expected that genetic changes are involved in the development of progesterone insensitivity in endometrial carcinomas. We therefore started an investigation to identify, isolate and characterise progesterone-regulated genes involved in progesterone-induced growth inhibition in endometrial carcinoma cells. Using differential display PCR eight progesterone-regulated cDNA clones were identified in endometrial carcinoma cell lines. Four of these progesterone-regulated cDNA clones were regulated in the for growth progesterone-sensitive cell line IK-3H12 and not regulated in the for growth-insensitive cell line ECC-1. This indicates that these four cDNA clones represent potentially important genes, which could be involved in inhibition of growth of endometrial carcinoma tissue by progesterone.     

Effect of levonorgestrel and mifepristone on endometrial receptivity markers in a three-dimensional human endometrial cell culture model

OBJECTIVE: To investigate the effect of levonorgestrel and mifepristone on the expression of endometrial receptivity markers in a three-dimensional endometrial construct. DESIGN: In vitro study. SETTING: University hospital and research laboratory. PATIENT(S): Twelve fertile donors. INTERVENTION(S): Timed endometrial biopsy. MAIN OUTCOME MEASURE(S): Examine the effect of levonorgestrel along with another well-studied fertility-regulating drug, mifepristone, on the expression of endometrial receptivity factors in a three-dimensional stromal and epithelial cell coculture model by immunohistochemistry. RESULT(S): Both epithelial and stromal cells of in vitro endometrial construct showed the presence of estrogen receptor-alpha, estrogen receptor-beta, progesterone receptors-(A+B), vascular endothelial growth factor, leukemia inhibitory factor, interleukin-1 beta, and cyclooxygenase-2, whereas the expression of progesterone receptor-B (AR), integrin alpha(V)beta(3,) and MUC1 were confined to epithelial cells. Mifepristone up-regulated expression of epithelial estrogen receptor-beta and progesterone receptor-B and down-regulated stromal vascular endothelial growth factor and surface molecules MUC1 and integrin alpha(V)beta(3) as observed in vivo. Levonorgestrel had no effect on the expression of endometrial receptivity markers studied. CONCLUSION(S): This in vitro model expresses progesterone-regulated endometrial receptivity factors seen in the physiologic condition. Treatment with levonorgestrel did not affect the expression of these endometrial receptivity markers in contrast to mifepristone. This in vitro model holds the potential to study endometrial receptivity, the embryo-endometrial interaction, and develop new agents for fertility control.     

子宫内膜癌组织中雌激素及孕激素受体亚型的表达研究

目的探讨子宫内膜癌组织中雌激素受体(ER)亚型mRNA及孕激素受体(PR)亚型mRNA和蛋白表达水平的变化及其意义。方法采用RT-PCR法检测66例子宫内膜癌和30例正常子宫内膜组织ER、PR亚型mRNA的表达,采用蛋白印迹法检测PR亚型蛋白的表达。结果(1)ERαmRNA在子宫内膜癌和正常子宫内膜组织中的表达水平分别是8.00±7.77、3.84±2.57,而ERβmRNA的表达水平分别是4.15±3.55、0.41±0.29,子宫内膜癌组织中ERα、ERβmRNA表达水平均高于正常子宫内膜,两种组织间分别比较,差异均有统计学意义(P<0.05)。(2)PR、PRA和PRB蛋白表达及PR mRNA表达的降低与子宫内膜癌的发生有关(P<0.05),而PRA与PRB蛋白表达的比值和PRB mRNA的表达与子宫内膜癌的发生无明显相关性(P值分别为0.550、0.901)。(3)子宫内膜癌组织中PRB mRNA与ERβmRNA的表达水平呈正相关关系(r=0.43,P<0.01)。结论子宫内膜癌组织中ER亚型mRNA表达上调、PR蛋白和mRNA表达的下调可能参与了子宫内膜癌的发生;PRB mRNA与ERβmRNA表达呈正相关关系。     

大剂量米非司酮和孕激素联合应用治疗子宫内膜癌的临床研究

目的　研究大剂量米非司酮、大剂量孕激素以及二者联合应用对子宫内膜癌患者的治疗效果 ,并探讨其作用机制。方法　将 3 0例经诊刮确诊的子宫内膜癌患者随机分为 3组 ,每组 10例 ,术前用药 5d。米非司酮组术前用米非司酮 ( 10 0mg d) ,醋酸甲羟孕酮组术前用醋酸甲羟孕酮 ( 50 0mg d) ,联合组术前用米非司酮 ( 10 0mg d)、醋酸甲羟孕酮 ( 50 0mg d)。各组用药前后行自身对照 ,观察癌细胞组织形态学改变及雌激素受体 (ER)、孕激素受体 (PR)、增殖细胞核抗原 (PCNA)、凋亡相关基因 (bcl 2、bax)及CD44 v6基因表达的变化。结果　各组治疗后 ,癌细胞分化均趋向成熟 ,分泌活跃 ,可在癌组织中观察到凋亡的癌细胞 ,联合组治疗后变化最明显。醋酸甲羟孕酮组治疗前后 ,免疫组织化学 (免疫组化 )评分分别为PR( 2 9± 1 1、1 6± 0 8)、ER( 2 8± 0 9、1 4± 0 9)、PCNA( 0 84± 0 11、0 60±0 12 )、bcl 2 ( 0 2 3 6± 0 0 89、0 157± 0 981)和CD44 v6( 4 6± 1 8、2 5± 1 9) ,表达均降低 (所有P <0 0 1) ,bax( 0 2 0± 0 10、0 42± 0 0 7)表达增多 (P <0 0 1)。米非司酮组治疗前后免疫组化评分分别为PR( 3 4± 1 0、1 9± 0 8)、ER( 2 7± 0 9、1 2± 0 7)、PCNA( 0 80± 0 15、0